Summary Mast cells (MCs) are granulated tissue-resident cells of hematopoietic lineage which contribute to innate immunity but are best known for their roles in allergic diseases such as anaphylaxis, rhinitis, asthma, chronic urticaria and atopic dermatitis. In addition to the high affinity IgE receptor (Fc?RI), MCs express numerous G protein coupled receptors (GPCRs), which are the largest group of membrane receptor proteins known and are the most common targets of drug therapy. A recent exciting development in MC research has been the realization that a diverse group of cationic amphipathic peptides/proteins including antimicrobial host defense peptides (HDPs), neuropeptides (NPs), eosinophil granule proteins and many FDA approved peptidergic drugs all activate human MCs via a novel GPCR known as Mas-related gene-X2 (MrgX2). However, the molecular mechanisms involved in the activation/regulation of MrgX2 remain unknown. The hypothesis to be tested in this proposal is that HDPs, NPs and eosinophil granule proteins activate MrgX2 located in three different MC compartments and contribute to rosacea and chronic asthma. We have identified 42 naturally occurring missense variants of MrgX2 in the NHLBI Grand Opportunity Exome Sequencing Project (NHLBI GO ESP) database, which contains sequencing data from NHLBI's Severe Asthma Research Program (SARP) . In aim 1, selected variants (based on allele frequency) will be transfected in a MC line, RBL-2H3 cells and the ability of ligands (HDPs, NPs and eosinophil granule proteins) to induce signaling and degranulation will be determined. The domains of MrgX2 that interact with specific G proteins (G?q and G?i3) will be identified and the impact of this interaction on signaling and mediator release will be determined. Fluorescence lifetime imaging (FLIM) technology will be utilized to determine the mechanisms of MrgX2/G protein interaction in real time. In aim 2, we will test the hypothesis that MrgX2-mediated MC degranulation requires the integration of signaling input from the receptor in three compartments (cell surface, granules and lipid rafts). We will determine the roles of Orai channels, granule-associated autophagy proteins and lipid rafts on MC degranulation. The impact of modulating each of these signaling pathways on MrgX2- mediated MC degranulation will be determined. In aim 3, three in vivo models; (a) MrgprB2-/- mice (mouse ortholog of human MrgX2), (b) replacement of MrgprB2 with MrgX2 and (c) humanized mice that express MrgX2 in MCs will be used to determine the role MgX2 on experimental rosacea and chronic asthma. The effects of structural modification of MrgX2 and modulation of its signaling on rosacea and chronic asthma will be determined. Completion of this study will lead to a better understanding MrgX2 structure/function regulation in vitro and the modulation of chronic skin and lung diseases (rosacea and asthma) in vivo.